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Assessment of simple marker-free genetic transformation techniques in alfalfa.
- Source :
- Plant Cell Reports; Nov2011, Vol. 30 Issue 11, p1991-2000, 10p
- Publication Year :
- 2011
-
Abstract
- Methods to avoid the presence of selectable marker genes (SMG) in transgenic plants are available but not implemented in many crop species. We assessed the efficiency of simple marker-free Agrobacterium-mediated transformation techniques in alfalfa: regeneration without selection, or marker-less, and co-transformation with two vectors, one containing the SMG and one containing a non-selected gene. To easily estimate the efficiency of marker-less transformation, the nptII and the GUS markers were used as non-selected genes. After Agrobacterium treatment, somatic embryos were regenerated without selection. The percentage of transgenic embryos was determined by a second cycle of regeneration using the embryos as starting material, in the presence of kanamycin, by PCR screening of T1 progenies, and by the GUS test. In two experiments, from 0 to 1.7% of the somatic embryos were transgenic. Co-transformation was performed with two vectors, one with the hemL SMG and one with the unselected nptII gene, each carried by a different culture of Agrobacterium. Only 15 putative co-transformed plants were regenerated from two experiments, with an average co-transformation percentage of 3.7. Southern blot hybridizations and/or T progeny segregation were used to confirm transgene integration, and qPCR was also used to estimate the T-DNA copy number. In the T progenies obtained by crossing with a non-transgenic pollinator, marker-free segregants were obtained. Both marker-free approaches showed very low efficiency. [ABSTRACT FROM AUTHOR]
Details
- Language :
- English
- ISSN :
- 07217714
- Volume :
- 30
- Issue :
- 11
- Database :
- Complementary Index
- Journal :
- Plant Cell Reports
- Publication Type :
- Academic Journal
- Accession number :
- 66548953
- Full Text :
- https://doi.org/10.1007/s00299-011-1107-x